A method for preparing a material carrying vancomycin

Abstract

The invention relates to the field of vancomycin research technology, and discloses a method for preparing vancomycin-carrying materials, including the preparation of vancomycin-carrying nanodiamonds, the preparation of carboxyl nanodiamonds, and the preparation of vancomycin-carrying nanodiamond-45S5 bioglass composite tissue engineering bone; the nanodiamonds are subjected to ultrasonic treatment, and functional modification is performed by an improved strong acid oxidation method to expose a large number of carboxyl functional groups on their surfaces, which are covalently bonded with the hydroxyl functional groups of vancomycin through ester bonds to prepare nanodiamonds carrying vancomycin. The vancomycin-carrying nanodiamond-45S5 bioglass composite tissue engineering bone is implanted into the infected bone defect site, and while the infected bone defect is locally filled, vancomycin is slowly released, common infectious bone defect pathogens are inhibited, and its osteogenic and angiogenic activity is exerted to promote the repair and healing of the infected bone defect, providing a new idea and approach for the repair and treatment of the infected bone defect.

Description

Technical Field

[0001] The invention relates to the technical field of vancomycin-loaded research, and in particular to a method for preparing a vancomycin-loaded material. Background Art

[0002] Vancomycin is a glycopeptide macromolecular antibiotic, and its mechanism of action is mainly to inhibit the synthesis of cell walls by interfering with peptidoglycan, an important component in the bacterial cell wall structure, and inhibit the production of phospholipids and polypeptides in the cell wall to exert a bactericidal effect. Vancomycin has a killing effect on Gram-positive rods and cocci such as Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae, which are common in infected bone defects, and has an excellent bactericidal effect on methicillin-resistant Staphylococcus aureus, which has extremely strong drug resistance. However, systemic application of vancomycin is prone to cause toxic side effects such as hearing loss, respiratory depression and even liver and kidney damage. With the rapid development of mechanization and automation in agriculture, construction, manufacturing and transportation, the occurrence of malignant accidents and the treatment of various types of open fractures and bone defects caused by them have become increasingly important. Open fractures caused by severe high-energy trauma often leave residual foreign matter and are accompanied by nerve and vascular damage. It is difficult to achieve complete and thorough debridement in the early stage. The bacteria in the residual infection focus will gradually spread to the entire bone tissue to form acute or chronic osteomyelitis, which brings great difficulties to subsequent treatment. Suppurative osteomyelitis caused by prolonged unhealed open fractures is the most dangerous infectious bone disease among osteomyelitis. It is a serious complication after fracture. The infection lesions are often located at the fracture site and gradually spread to both ends of the bone tissue. In the later stage, dead cavity is often formed and abscesses in the surrounding soft tissues are caused. The dead cavity and abscesses rupture to form sinuses. At the same time, bone necrosis and nonunion occur repeatedly for a long time, eventually forming infected bone defects. Severe cases may even cause systemic infection and endanger the patient's life, thereby increasing the difficulty of healing. Summary of the invention

[0003] 1. Technical issues to be resolved

[0004] In view of the deficiencies of the prior art, the present invention provides a method for preparing a vancomycin-loaded material, which has the advantages of providing new materials and approaches for the treatment of infected bone defects, and solves the problem of composite tissue engineering bone treatment of infected bone defects.

[0005] (II) Technical solution

[0006] To achieve the above object, the present invention provides the following technical solutions: a method for preparing a material carrying vancomycin, including the preparation of nanodiamonds carrying vancomycin, the preparation of carboxyl nanodiamonds, and the preparation of vancomycin-nanodiamond-45S5 bioglass composite tissue engineering bone;

[0007] The nanodiamond is subjected to ultrasonic treatment and functionalized by an improved strong acid oxidation method to expose a large number of carboxyl functional groups on its surface, which are then covalently bonded with the hydroxyl functional groups of vancomycin through ester bonds to prepare nanodiamond loaded with vancomycin.

[0008] Preferably, the preparation of the carboxyl nanodiamond comprises the following steps:

[0009] S101, weigh 1.2 g of nano-diamond powder and dissolve it in 150 ml of deionized water, centrifuge at 3500 rpm, and discard the lower precipitate;

[0010] S102, placing the upper nano-diamond suspension in a small beaker and freeze-drying it for 24 hours to obtain nano-diamond powder with finer particles;

[0011] S103, weighing 200 mg of the nano-diamond powder with finer particles and adding it into a flask containing 25 mL of a mixed acid solution (the volume ratio of concentrated sulfuric acid to concentrated nitric acid is 3:1);

[0012] S104, after 24 hours at 55°C, centrifuge at 10,000 rpm, discard the supernatant, and heat the lower precipitate in NaOH (0.1 mol / L) and HCl (0.1 mol / L) at 85°C for 2.5 hours;

[0013] S105, centrifuging at 10000 rpm, discarding the supernatant to obtain carboxyl nanodiamonds, and washing the carboxyl nanodiamonds with deionized water 10 times to make them nearly neutral;

[0014] S106, centrifuge at 3000 rpm, discard the lower precipitate, and freeze-dry the upper carboxyl nano-diamond suspension for 24 hours to obtain finer carboxyl nano-diamond powder for later use.

[0015] Preferably, the preparation of the vancomycin-loaded nanodiamond comprises the following steps:

[0016] S201, weigh 120 mg of carboxyl nanodiamond into a flask, add SOCl 2 After 25 mL of the solution was dissolved by ultrasonication, 1.2 mL of DMF was added, ultrasonication was performed at 45 °C for 3 h, and heating was performed at 75 °C under reflux for 24 h;

[0017] S202, after the reaction is completed, the reaction mixture is centrifuged at 10000 rpm, the lower precipitate is washed 5 times with dry THF, and vacuum dried at 30°C to obtain carboxyl nanodiamond-COCl;

[0018] S203, weighing 120 mg of carboxyl nanodiamond-COCl and 350 mg of vancomycin respectively and placing them in a flask;

[0019] S204, add 25 mL of DMF and dissolve by ultrasonication, then add 1 mL of triethylamine under stirring at room temperature;

[0020] S205, centrifuging at 10000 rpm, then washing the lower precipitate twice with anhydrous ethanol, and vacuum drying the precipitate obtained by centrifugation at 50°C for 24 hours to obtain vancomycin-loaded nanodiamonds;

[0021] In the step S204, ultrasonic treatment is performed at 40° C. for 4 hours, and the reaction is performed at 60° C. for 2 days.

[0022] Preferably, the preparation of the vancomycin-loaded-nanodiamond-45S5 bioglass composite tissue engineering bone comprises the following steps:

[0023] S301. Using computer-aided design (CAD) to design a scaffold model of composite tissue engineering bone;

[0024] S302, using polyhydroxybutyric acid hexanoate (PHBHHx) as a binder in 3D printing to bond drug-loaded powder and bioceramics;

[0025] S303, dissolving PHBHHx powder in chloroform to form a polymer solution, adding a mixture of vancomycin-loaded nanodiamond and 45S5 bioactive glass to the solution, and stirring thoroughly to form a uniform paste;

[0026] S304, using the 4th generation 3D-Bioplotter system (EnvisionTEC, Germany) to print the prepared paste by applying compressed air to prepare a layered porous composite tissue engineering bone scaffold (φ=6 mm×h=8 mm, layer spacing 600 μm);

[0027] S305. Prepare vancomycin-nanodiamond-45S5 bioglass composite tissue engineering bones (VNB), freeze-dry the composite tissue engineering bones, and place them at -20°C for later use.

[0028] Preferably, the vancomycin-loaded-nanodiamond-45S5 bioglass composite tissue engineering bone uses CAD technology to design a three-dimensional tissue engineering bone model with a suitable pore structure, and the vancomycin-loaded nanodiamond is mixed with 45S5 bioglass with good biocompatibility, osteogenic activity and degradability. The vancomycin-loaded-nanodiamond-45S5 bioglass composite tissue engineering bone is prepared using micro-jet free-forming 3D printing technology that can work at room temperature, which can maximize the efficacy of vancomycin.

[0029] Compared with the prior art, the present invention provides a method for preparing a material carrying vancomycin, which has the following beneficial effects:

[0030] 1. The preparation method of the vancomycin-loaded material. Although the vancomycin-loaded tissue engineering material has been applied to the research of orthopedic diseases, the drug loading efficiency is not ideal. Compared with the existing drug-loaded sustained-release system (such as polylactic acid-glycolic acid, etc.), the surface-modified nanodiamond has a more efficient drug loading effect on vancomycin.

[0031] 2. The preparation method of the vancomycin-loaded material is to implant the vancomycin-nanodiamond 45S5 bioglass composite tissue engineering bone into the infected bone defect site, slowly release vancomycin while locally filling the infected bone defect, inhibit common infectious bone defect pathogens, and exert its osteogenic and angiogenic activity, aiming to promote the repair and healing of infected bone defects, and provide new ideas and approaches for the repair and treatment of infected bone defects.

[0032] 3. The preparation method of the vancomycin-loaded material can select the loaded drug according to the drug sensitivity results of the patient's infectious bone defect, and use bone tissue engineering technology, 3D printing technology and nanotechnology to quickly prepare personalized composite tissue engineering bones, carry out targeted and precise individualized treatment, and obtain definite treatment effects, which has important scientific research and clinical application significance. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] A method for preparing a material carrying vancomycin, including the preparation of nanodiamonds carrying vancomycin, the preparation of carboxyl nanodiamonds, and the preparation of vancomycin-nanodiamond-45S5 bioglass composite tissue engineering bone;

[0035] The nanodiamond is subjected to ultrasonic treatment and functionalized by an improved strong acid oxidation method to expose a large number of carboxyl functional groups on its surface, which are then covalently bonded with the hydroxyl functional groups of vancomycin through ester bonds to prepare nanodiamond loaded with vancomycin.

[0036] Furthermore, the preparation of the carboxyl nanodiamond comprises the following steps:

[0037] S101, weigh 1.2 g of nano-diamond powder and dissolve it in 150 ml of deionized water, centrifuge at 3500 rpm, and discard the lower precipitate;

[0038] S102, placing the upper nano-diamond suspension in a small beaker and freeze-drying it for 24 hours to obtain nano-diamond powder with finer particles;

[0039] S103, weighing 200 mg of the nano-diamond powder with finer particles and adding it into a flask containing 25 mL of a mixed acid solution (the volume ratio of concentrated sulfuric acid to concentrated nitric acid is 3:1);

[0040] S104, after 24 hours at 55°C, centrifuge at 10,000 rpm, discard the supernatant, and heat the lower precipitate in NaOH (0.1 mol / L) and HCl (0.1 mol / L) at 85°C for 2.5 hours;

[0041] S105, centrifuging at 10000 rpm, discarding the supernatant to obtain carboxyl nanodiamonds, and washing the carboxyl nanodiamonds with deionized water 10 times to make them nearly neutral;

[0042] S106, centrifuge at 3000 rpm, discard the lower precipitate, and freeze-dry the upper carboxyl nano-diamond suspension for 24 hours to obtain finer carboxyl nano-diamond powder for later use.

[0043] Furthermore, the preparation of the vancomycin-loaded nanodiamond comprises the following steps:

[0044] S201, weigh 120 mg of carboxyl nanodiamond into a flask, add SOCl 2 After 25 mL of the solution was dissolved by ultrasonication, 1.2 mL of DMF was added, ultrasonication was performed at 45 °C for 3 h, and heating was performed at 75 °C under reflux for 24 h;

[0045] S202, after the reaction is completed, the reaction mixture is centrifuged at 10000 rpm, the lower precipitate is washed 5 times with dry THF, and vacuum dried at 30°C to obtain carboxyl nanodiamond-COCl;

[0046] S203, weighing 120 mg of carboxyl nanodiamond-COCl and 350 mg of vancomycin respectively and placing them in a flask;

[0047] S204, add 25 mL of DMF and dissolve by ultrasonication, then add 1 mL of triethylamine under stirring at room temperature;

[0048] S205, centrifuging at 10000 rpm, then washing the lower precipitate twice with anhydrous ethanol, and vacuum drying the precipitate obtained by centrifugation at 50°C for 24 hours to obtain vancomycin-loaded nanodiamonds;

[0049] In the step S204, ultrasonic treatment is performed at 40° C. for 4 hours, and the reaction is performed at 60° C. for 2 days.

[0050] Furthermore, the preparation of the vancomycin-nanodiamond-45S5 bioglass composite tissue engineering bone comprises the following steps:

[0051] S301. Using computer-aided design (CAD) to design a scaffold model of composite tissue engineering bone;

[0052] S302, using polyhydroxybutyric acid hexanoate (PHBHHx) as a binder in 3D printing to bond drug-loaded powder and bioceramics;

[0053] S303, dissolving PHBHHx powder in chloroform to form a polymer solution, adding a mixture of vancomycin-loaded nanodiamond and 45S5 bioactive glass to the solution, and stirring thoroughly to form a uniform paste;

[0054] S304, using the 4th generation 3D-Bioplotter system (EnvisionTEC, Germany) to print the prepared paste by applying compressed air to prepare a layered porous composite tissue engineering bone scaffold (φ=6 mm×h=8 mm, layer spacing 600 μm);

[0055] S305. Prepare vancomycin-nanodiamond-45S5 bioglass composite tissue engineering bones (VNB), freeze-dry the composite tissue engineering bones, and place them at -20°C for later use.

[0056] Furthermore, the vancomycin-loaded-nanodiamond-45S5 bioglass composite tissue engineering bone uses CAD technology to design a three-dimensional tissue engineering bone model with a suitable pore structure, mixes the vancomycin-loaded nanodiamond with 45S5 bioglass with good biocompatibility, osteogenic activity and degradability, and uses micro-injection free-forming 3D printing technology that can work at room temperature to prepare the vancomycin-loaded-nanodiamond-45S5 bioglass composite tissue engineering bone, which can maximize the efficacy of vancomycin.

[0057] Working principle: The preparation method of the material loaded with vancomycin, this invention uses bone tissue engineering technology, 3D printing technology and nanotechnology to prepare efficient nanodiamond loaded with vancomycin and composite tissue engineering bone (Vancomycin-Nanodiamond-45S5Bioactive glass composite tissue engineering bone, VNB) using the drug-loaded nanomaterial, and implants the vancomycin-nanodiamond-45S5 bioactive glass composite tissue engineering bone into the infected bone defect site, while filling the infected bone defect locally, it slowly releases vancomycin, inhibits common infectious bone defect pathogens, and exerts its osteogenic and angiogenic activity, aiming to promote the repair and healing of infected bone defects, and provide new ideas and approaches for the repair and treatment of infected bone defects. In the long term, the loaded drugs can be selected according to the drug sensitivity results of the patient's infected bone defect, and personalized composite tissue engineering bones can be quickly prepared using bone tissue engineering technology, 3D printing technology, and nanotechnology, so as to carry out targeted and precise individualized treatment and obtain definite treatment effects, which has important scientific research and clinical application significance;

[0058] The vancomycin-nanodiamond-45S5 bioglass composite tissue engineering bone prepared by bone tissue engineering technology, 3D printing technology and nanotechnology can slowly release vancomycin while filling the infected bone defect locally, inhibit common pathogenic bacteria of infected bone defects, and exert its osteogenic and angiogenic activity, which can theoretically promote the repair and healing of infected bone defects. The research results of the present invention reveal the effect and mechanism of vancomycin-nanodiamond materials and vancomycin-nanodiamond-45S5 bioglass composite tissue engineering bone in treating infected bone defects, expand the research and application of various disciplines and technical resources such as bone tissue engineering technology, 3D printing technology and nanotechnology in bone infection-related diseases, and provide new materials and approaches for the treatment of infected bone defects.

[0059] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a material carrying vancomycin, characterized in that: Including the preparation of carboxyl nanodiamonds, the preparation of nanodiamonds loaded with vancomycin, and the preparation of vancomycin-nanodiamond-45S5 bioglass composite tissue engineering bone; The nanodiamond is subjected to ultrasonic treatment and functionalized by an improved strong acid oxidation method to expose a large number of carboxyl functional groups on its surface, and then covalently bonded with the hydroxyl functional groups of vancomycin through ester bonds to prepare nanodiamond loaded with vancomycin; The preparation of the carboxyl nanodiamond comprises the following steps: S101, weigh 1.2 g of nano-diamond powder and dissolve it in 150 ml of deionized water, centrifuge at 3500 rpm, and discard the lower precipitate; S102, placing the upper nano-diamond suspension in a small beaker and freeze-drying it for 24 hours to obtain nano-diamond powder with finer particles; S103, weighing 200 mg of the nano-diamond powder with finer particles and adding it into a flask containing 25 mL of a mixed acid solution of concentrated sulfuric acid and concentrated nitric acid in a volume ratio of 3:1; S104, after 24 hours at 55°C, centrifuge at 10,000 rpm, discard the supernatant, and heat the lower precipitate in 0.1 mol / L NaOH and 0.1 mol / L HCl at 85°C for 2.5 hours; S105, centrifuging at 10000 rpm, discarding the supernatant to obtain carboxyl nanodiamonds, and washing the carboxyl nanodiamonds with deionized water 10 times to make them nearly neutral; S106, centrifuge at 3000 rpm, discard the lower sediment, freeze-dry the upper carboxyl nano-diamond suspension for 24 hours, and obtain finer carboxyl nano-diamond powder for later use; The preparation of the vancomycin-loaded nanodiamond comprises the following steps: S201, weigh 120 mg of carboxyl nanodiamond into a flask, add 25 mL of SOCl2 and ultrasonically dissolve, then add 1.2 mL of DMF, ultrasonically dissolve at 45°C for 3 h, and heat under reflux at 75°C for 24 h; S202, after the reaction is completed, the reaction mixture is centrifuged at 10000 rpm, the lower precipitate is washed 5 times with dry THF, and vacuum dried at 30°C to obtain carboxyl nanodiamond-COCl; S203, weighing 120 mg of carboxyl nanodiamond-COCl and 350 mg of vancomycin respectively and placing them in a flask; S204, add 25 mL of DMF and dissolve by ultrasonication, then add 1 mL of triethylamine under stirring at room temperature; S205, centrifuging at 10000 rpm, then washing the lower precipitate twice with anhydrous ethanol, and vacuum drying the precipitate obtained by centrifugation at 50°C for 24h to obtain vancomycin-loaded nanodiamonds; In step S204, ultrasonic treatment is performed at 40° C. for 4 hours, and the reaction is performed at 60° C. for 2 days; The preparation of the vancomycin-loaded-nanodiamond-45S5 bioglass composite tissue engineering bone comprises the following steps: S301. Using computer-aided design (CAD) to design a scaffold model of composite tissue engineering bone; S302, using polyhydroxybutyric acid hexanoate PHBHHx as a binder in 3D printing to bond drug-loaded powder and bioceramics; S303, dissolving PHBHHx powder in chloroform to form a polymer solution, adding a mixture of vancomycin-loaded nanodiamond and 45S5 bioactive glass to the solution, and stirring thoroughly to form a uniform paste; S304, using the 4th generation 3D-Bioplotter system, EnvisionTEC, Germany, the prepared paste was printed by applying compressed air to prepare layered porous composite tissue engineering bone scaffolds, φ = 6 mm × h = 8 mm, and the layer spacing was 600 μm; S305. Prepare the vancomycin-loaded-nanodiamond-45S5 bioglass composite tissue engineering bone, freeze-dry the composite tissue engineering bone, and place it at -20°C for later use.

2. The method for preparing a vancomycin-loaded material according to claim 1, characterized in that: The vancomycin-loaded-nanodiamond-45S5 bioglass composite tissue engineering bone uses CAD technology to design a three-dimensional tissue engineering bone model with a suitable pore structure, mixes the vancomycin-loaded nanodiamond with 45S5 bioglass with good biocompatibility, osteogenic activity and degradable performance, and uses micro-jet free-forming 3D printing technology that can work at room temperature to prepare the vancomycin-loaded-nanodiamond-45S5 bioglass composite tissue engineering bone, thereby maximizing the efficacy of vancomycin.